Regulation of EMT During Avian Heart Valve Formation

描述（由申请人提供）：在心脏发育过程中，由于TGFβ介导的上皮间质细胞转化（EMT），瓣膜祖细胞首先在房室管中出现。此前的研究表明，EMT可分为两个阶段，激活阶段和侵袭阶段。第 1 阶段部分由转化生长因子 β 2 (TGF-2) 介导，第 2 阶段部分由 TGF-3 介导。先前的研究表明，EMT 可以通过抑制 5 种不同的 TGFβ 受体、TGFβ 受体 II、TGFβ 受体 III、Endoglin、ALK2 和 ALK5 来破坏。我们的假设是，还有两个独立的信号复合物介导 TGF 调节的 EMT。为了继续分析 TGFβ 介导的正常心脏发育调节，首要目标是检查特定 TGFβ 亚型与介导 EMT 调节的受体之间的相关性。将进行微阵列实验来评估特定TGFβ亚型的损失对EMT之前、EMT期间以及EMT之后的间充质细胞中的组织中基因表达改变的影响。然后，TGFβ调节基因中选定的标记将用于评估基因表达是否随着每个特定受体被破坏而改变。这些数据之间的相关性将指向介导 EMT 的特定受体和配体信号复合物。这些假定的复合物将在蛋白质水平上得到证实。其他实验室的数据表明心脏 EMT 中涉及多种信号转导机制和细胞内调节因子，包括 ErbB2、NF-1、VEGF 和 NFATc1。在第二个目标中，将进行实验以确定TGFβ信号传导的破坏是否会改变任何其他候选机制，或者这些分子的丢失是否会改变TGFβ配体或受体的表达。在神经嵴系统中观察到，在神经管中强制表达Sox 8、9或10会引起EMT并产生异位神经嵴细胞。我们推测这类似于心脏中的细胞入侵步骤，并且它需要一些 TGF¿ 活性。第三个目标是通过实验来确定 Sox 基因的外源表达是否会强制 EMT，以及 Sox 基因表达是否受 TGF 信号转导调节或调节 TGF 介导的反应。这些实验将为 EMT 调节机制提供新的线索，并有助于了解先天性心脏病。

DESCRIPTION (provided by applicant): During cardiac development, valve progenitors first arise in the atrioventricular canal as a result of a TGF¿-mediated epithelial-mesenchymal cell transformation (EMT). Prior studies showed that this EMT can be divided into two stages, an activation stage and an invasion stage. Stage 1 is mediated, in part, by transforming growth factor beta 2 (TGF¿2) and stage 2 is mediated, in part, by TGF¿3. Previous work showed that EMT can be disrupted by the inhibition of 5 different TGF¿ receptors, TGF¿ receptor II, TGF¿ receptor III, Endoglin, ALK2 and ALK5. It is our hypothesis that there are two more discrete signaling complexes mediating TGF¿-regulated EMT. To continue the analysis of TGF¿-mediated regulation of normal heart development, the first aim will examine the correlation between specific TGF¿ isoforms and receptors in mediating EMT regulation. Microarray experiments will be performed to assess the effect loss of specific TGF¿ isoforms on altered gene expression in tissues prior to EMT, during EMT and in mesenchymal cells after EMT. Selected markers among the TGF¿-regulated genes will then be used to assess whether gene expression is altered as each of the specific receptors is disrupted. Correlations between these data will point to specific receptor and ligand signaling complexes that mediate EMT. These putative complexes will be confirmed at the protein level. Data from other labs has implicated a variety of signal transduction mechanisms and intracellular regulators in cardiac EMT including ErbB2, NF-1, VEGF and NFATc1. In the second aim, experiments will be undertaken to determine whether disruption of TGF¿ signaling alters any of the other candidate mechanisms or whether loss of these molecules alters TGF¿ ligand or receptor expression. It was observed in the neural crest system that forced expression of Sox 8, 9 or 10 in the neural tube would cause an EMT and produce ectopic neural crest cells. We conjecture that this is analogous to the cell invasion step in the heart and that it requires some TGF¿ activity. In the third aim, experiments will be undertaken to determine whether exogenous expression of Sox genes will force EMT and whether Sox gene expression is regulated by TGF¿ signal transduction or regulates TGF¿-mediated responses. Together these experiments will shed new light on the mechanisms of EMT regulation and aid in an understanding of congenital heart disease.